Project Summary We seek to understand the structure of human papillomavirus (HPV) genomes in head and neck cancers by continuing our analysis of DNA sequence data from The Cancer Genome Atlas. We hypothesize that the type of structure of the viral genome contributes to disease outcome. Previous studies of HPV cancers have broadly assumed three viral genome structures; episomal, integrated, a mixture of both. We present evidence that the situation is more complex, with the possibility of viral-host DNA episomes. This research is critical because the incidence of HPV+ oropharyngeal cancer (OPC) is increasing and is currently considered an epidemic; recent reports have shown that 80% of these tumors are HPV+; HPV16 is present in 90% of these. While HPV+ OPC patients respond better to chemoradiation and have a ~60% higher 5 year survival rate than those who are HPV negative, there is a subset of ~20% HPV+ patients that do not respond well to therapy and/or suffer from cancer recurrence who should not receive de-escalated treatment; identifying these individuals is a priority so that they are not under treated. The questions we are posing and will answer are: How often does the HPV genome integrate into the human genome and excise with human DNA to reform a new episome? We have made several observations from our preliminary studies and one relevant to this question is that the episomal HPV genome can integrate into the human genome and subsequently excise to form a new episome with HPV and human DNA. This autonomously replicating molecule can therefore amplify not only intact viral oncogenes E6 and E7 but also human genes/sequences that may contribute to cancer development. Is the excised HPV structure or other structures of the HPV genome in HPV+ OPC a predictor of disease outcome? Based on the determined structures of the HPV genome in the samples available, we will preliminarily investigate if patients with viral-human episome-containing samples have a worse disease outcome. Are in vitro HPV16 transformed tonsil cells a model for studying HPV+ OPC? We have already identified hybrid viral-human DNA episomes present in some of the TCGA HPV positive HNSCC data. We would like to model some of these structures in primary tonsil cells. We propose to first analyze gene expression from tonsil cells transformed with HPV16 using RNA-seq. We will determine a) if the expression of the viral genome in these cells is similar to the patterns observed in the HPV+ OPC and b) if the host gene expression pattern is similar to that of HPV+ OPC. If the answer to these questions is yes then further studies of these cells as a model for HPV+ OPC transformed cells would be warranted, including transfecting into cells some of the aberrant HPV16-host genome structures reported in this application.